Thegoaloftheproposedresearchistogenerategeneticallyencodedbioluminescenttagsforlive-cell luminescence-basedphotoactivatedlocalizationmicroscopy(L-PALM).Thisrevolutionarymodeof superresolutionimagingwillmaintainallofthebenefitsoffluorescencePALM(fPALM)butwilleliminatethe needforexcitationlight.fPALMislargelyunsuitableforimaginglivecellsbecauseitrequireshighexcitation intensitiesthatleadtophototoxicity.Becauseluminescencegenerateslightwithouttheneedforexternal excitation,L-PALMwillnotsufferfromthislimitation.Thelatestgenerationofgeneticallyencoded bioluminescentlabelsarewellsuitedforwidefieldmicroscopyofsubcellularstructures,butarestill approximately1000-foldtoodimtobeusedforsingle-moleculelocalizationonpracticaltimescales.Toremedy thisdeficiency,thisstudyisdesignedtoproducebioluminescentprobeswithphotonoutputrates sufficienttolocalize~100,000moleculesinoneminute.Togeneratethisincreasedoutput,luciferaseswill firstbecoupledtoourbrightestfluorescentproteinstomaximizeluminescencequantumyieldviatheFrster resonanceenergytransfermechanism.Oncemaximaloutputisachievedinthisfirststep,theluciferase portionofthefusionwillthenbesubjectedtostructure-guideddirectedevolutiontargetedatlowering oxyluciferinbindingaffinityandthusincreasingthecatalyticrateoftheenzyme.Suchalterationsarepredicted toreducetheluminescencequantumyieldoftheluciferase,butenergytransfertoafluorescentproteinwill rescuetheluminescence,allowingmuchfasterenzymestobeengineeredwiththisstrategy.Tobeusefulfor live-cellL-PALM,bioluminescentprobesmustalsobecapableofswitchingonandoffcontrollablyto preventsignaloverlapbetweenindividualmoleculesineachimageframe.Twoindependentmechanismsfor producingswitchablelightoutputwillbepursuedinthisproject:(1)optimizationofenergytransferbetween luciferasesandphotoswitchablefluorescentproteins,followedbydirectedevolutiontoincreaselightoutput andimproveswitchingkinetics;?(2)insertionoflight-modulateddomainsintosplitluciferasesinorderto allostericallycontrolenzymeactivity.Throughouttheproject,heavyemphasiswillbeplacedonRosetta-based structure-guidedcomputationaldesignforgeneratingnovelluciferase-fluorescentproteinfusiontopologies, alteringluciferaseactivesiteenvironments,andengineeringallosterically-regulatedluciferases.Directed evolutionwithimage-basedscreeningwillthenbetheprimaryapproachforimprovingthepropertiesofprobes underdevelopmentineachaim.Theendproductsofthisprojectwillbeasetofgeneticallyencoded bioluminescentprobeswithbrightnessandphotoswitchingpropertiessuitableforthedevelopmentofL- PALMmethodologies.BeyondtheirultimateutilityforL-PALMimaging,manyoftheprobescreatedinthe courseofthisprojectwillbethebrightestandhighest-performingbioluminescenttagsyetdeveloped,andas suchwillhighlyusefulinnumerousotherlive-cellandwhole-organismimagingapplications.